The yeast Yarrowia lipolytica efficiently utilizes various hydrophobic substrates such as n-alkanes and fatty acids as a sole carbon source. Because of these properties and qualification as a GRAS (Generally Regarded As Safe) organism by FDA, it has been a target of intensive study for industrial application and used as a model organism to study the metabolism of hydrophobic substrates and its regulation.

In Y. lipolytica, n-alkanes are metabolized to fatty acids through sequential terminal oxidation initiated by cytochromes P450 (P450ALKs). Fatty acids synthesized endogenously or incorporated from culture media are activated to fatty-acyl CoA and metabolized by β-oxidation in peroxisome. Transcription of ALK1, a primary P450ALK, is induced by n-alkanes. It is positively regulated by the hetero-complex composed of two basic helix-loop-helix (bHLH) transcription factors, Yas1p and Yas2p, via Alkane Responsive Element 1 (ARE1) and negatively by Yas3p. Likewise, transcription of a number of genes involved in the metabolism of fatty acids is activated in the presence of fatty acids. However, the molecular mechanism of the transcriptional activation in response to fatty acids has been concealed in Y. lipolytica. The aim of this study is to elucidate the mechanism of transcriptional regulation by fatty acids in Y. lipolytica.

1. Search for genes involved in the fatty acid utilization in Y. lipolytica

In Fungi, the fatty acid utilization is transcriptionally regulated by transcription factors of two distinct families. One is Oaf1p-Pip2p system, most extensively characterized in the yeast Saccharomyces cerevisiae, in which the Zn2Cys6 transcription factors, Oaf1p and Pip2p, together with the C2H2 zinc finger protein Adr1p, regulate the transcription of genes in response to fatty acids. An alternative is FarA system, originally reported in the filamentous fungus Aspergillus nidulans, in which FarA, belonging to another Zn2Cys6 transcription factor family, activates the transcription of genes involved in fatty acid utilization.

Orthologs of OAF1 of S. cerevisiae were searched in Y. lipolytica genome database. Translation products of YALI0F13321g, YALI0D10681g, and YALI0D17988g exhibited similarity with Oaf1p only in their zinc cluster domains at 38, 45 and 38% identities, respectively. Deletion mutants of these genes, Δyali0f13321g, Δyali0d10681g, Δyali0d17988g, Δyali0f13321gΔyali0d10681g, and Δyali0d17988gΔyali0d10681g, were constructed and their growth on oleic acid was analyzed. However, no growth defect was observed, in marked contrast to S. cerevisiae, in which deletion of OAF1 or PIP2 causes a severe growth defect on oleic acid. These results indicated that fatty acid utilization of Y. lipolytica is regulated by a mechanism distinct from that of S. cerevisiae.

Next, a deletion mutant of YALI0D126281g, an ortholog farA of A. nidulans was constructed and its growth was analyzed. Δyali0d126281g strain grew normally on glycerol, glucose, n-tetradecane, and n-hexadecane. Δyali0d12628g also grew on n-decane and n-dodecane with slight growth impairments. In contrast, it exhibited severe growth defects on lauric acid and myristic acid. The growth of Δyali0d12628g on oleic acid was partially impaired. The growth defects of the Δyali0d12628g strain on fatty acids were complemented by introduction of YALI0D12628g with its native promoter on a low copy plasmid. These results suggest that YALI0D12628g plays an important role in fatty acid utilization in Y. lipolytica. Thus, this gene was designated as POR1 (Primary Oleate Regulator 1).

2. Characterization of Por1p

The RLM-RACE analysis suggested that an open reading frame of POR1 is 2751 bp in size and encodes a 916-amino acid protein. Two introns of 80 bp and 90 bp were found in POR1. Por1p has three structural features: a fungal Zn2Cys6 binuclear cluster domain (amino acids 32 to 71), a fungal specific transcription factor domain (amino acid 327 to 422), and a glutamine-rich (Q-rich) domain (amino acid 661 to 742). The first and second domains are conserved in orthologs of FarA. The Q-rich domains were found in Ctf1p of Candida albicans and an ortholog in Neurospora crassa, but not in FarA and FarB of A. nidulans.

As shown previously, PAT1, POT1, POX2 and PEX5, which are involved in β-oxidation or peroxisome proliferation, were induced by oleic acid. However, the expression of these genes was distinctly diminished in the Δpor1 strain grown on oleic acid. POR1 was expressed on all carbon sources in the wild-type strain. In agreement with the results of northern blot analysis, the reporter b-galactosidase activities under the control of PAT1 promoter were increased by the growth on oleic acid and n-decane in the wild-type cells. In contrast, reporter activities in the Δpor1 strain were much less induced on oleic acid and partially on n-decane. These results suggest that Por1p functions in the transcriptional activation in response to fatty acids.

Next, the localization of Por1p fused with GFP at its C-terminus, expressed from a low copy plasmid under the control of its promoter, was observed in living cells. The fluorescent signal was enriched in the nuclear periphery in cells grown on glucose. Interestingly, Por1p-EGFP was also observed as one or a few spots at the nuclear periphery in live cells and this punctuate signals were more prominent in cells grown on oleic acid. When cells were fixed, fluorescent signal was observed irrespective of carbon sources. These results suggested that Por1p localizes to the periphery of inner nuclear membrane.

The deletion analysis of PAT1 promoter using lacZ as a reporter gene was performed to identify a DNA element responsible for the transcriptional regulation by Por1p. The results suggested that the sequence CGAGCCGA at -173 to -166 in the PAT1 promoter is important for the transcriptional activation by Por1p in response to fatty acid.

3. Other mechanisms involved in the regulation of fatty utilization in Y. lipolytica

The deletion mutant of POR1 exhibited weak growth on oleic acid. In addition, slight transcriptional activation of POT1, PAT1, POX1, and PEX5 by oleic acid was observed in the Δpor1 cells. These results indicated the presence of another mechanism(s) for the transcriptional activation by oleic acid in Y. lipolytica.

Transcription of genes involved in fatty acid utilization is repressed by glucose, inconsistent to ALK1, transcription of which is not repressed by glucose. The reporter assay using PAT1 promoter in the cells cultured on glucose, oleic acid, and glucose with oleic acid suggested that derepression from glucose significantly contribute the expression of PAT1 in the presence of oleic acid.

The ortholog of ADR1 of S. cerevisiae was searched in Y. lipolytica genome database and YALI0D18678g deduced to encode a protein belonging to the member of C2H2 Zinc finger protein was identified. This gene was designated as CFU1, (Control of Fatty acid Utilization 1). The reporter activity under the control of PAT1 promoter was decreased in Δcfu1 and Δpor1Δcfu1 on oleic acid and n-decane. These results suggested that the Cfu1p is involved in transcriptional regulation of PAT1 together with Por1p.

4. Conclusion

In this study, two genes, POR1 and CFUI, in involvement of the transcriptional regulation of fatty acid utilization in Y. lipolytica were identified and characterized. Although the mechanisms of fatty acid sensing and glucose derepression still remain unclear, this study will contribute to the understanding of entire regulatory mechanism of fatty acid utilization in Y. lipolytica.

Yarrowia lipolyticaはn-アルカン及び脂肪酸などの疎水性基質を効率よく利用して生育する酵母である。この酵母はクエン酸生産生物として米国FDAよりGRAS (Generally Regarded As Safe) の認定を受けており、n-アルカン及び脂肪酸などに由来する様々な産業上有用な物質の生産微生物として期待され、これら疎水性物質の代謝のしくみについて研究が進められている。

本酵母におけるn-アルカンの末端を水酸化するチトクロームP450ALKの制御については、転写活性化因子2種と抑制因子1種を含めてかなり詳細が判明しつつある。一方で、脂肪酸の利用に関わる遺伝子の発現の調節に関しては未解明であった。本論文は脂肪酸によって誘導される、パーオキシソーム及びβ酸化系を構成する様々な遺伝子に共通する転写調節因子の探索を行い、得られた候補遺伝子の性質を調べたものである。本論文は5章よりなり、第1章の序論を受け、第2章から第4章において研究の成果が述べられている。

第2章では、酵母Saccharomyces cerevisiaeにおいてヘテロ複合体を形成して脂肪酸に応答して転写活性化を行う、互に高い相同性を有するOaf1pとPip2pのY. lipolyticaにおけるオルソログ遺伝子を求めてY. lipolyticaゲノムデータベースを検索した。OAF1に対してZn2Cys6領域近傍においてのみ、それぞれ38%、45%、及び38%のアミノ酸相同性を有するYALI0F13321g、YALI0D10681g、及びYALI0D17988gを得た。これら遺伝子についての単独あるいは二重の遺伝子破壊株はオレイン酸を炭素源とする培地で正常に生育し、本酵母にはOAF1やPIP2に相当する遺伝子は存在しないものと推定された。そこで、糸状菌Aspergillus nidulansのZn2Cys6型転写因子をコードし、この菌の脂肪酸による生育に必要とされるfarAのオルソログの探索を行い、全領域にわたって高い相同性を有すYALI0D126281gを得た。この遺伝子の破壊株はグリセロール、グルコース、n-テトラデカン、n-ヘキサデカンを炭素源とする培地で正常に生育したが、ラウリン酸、ミリスチン酸を含む培地で生育せず、オレイン酸でも生育が制限されていた。そこで、この遺伝子をPOR1 (Primary Oleate Regulator 1)と命名した。

第3章では、POR1の性質について解析を行っている。RLM-RACE解析から、POR1は2個のイントロンを有し、916アミノ酸のタンパク質をコードしていた。 Por1pはアミノ酸32から71の間に菌類の転写因子に特有なZn2Cys6 binuclear cluster domainを有していた。β酸化系のチオラーゼ遺伝子PAT1とPOT1、アシルCoAオキシダーゼ遺伝子POX2、及びパーオキシンPEX5の脂肪酸存在下における発現をノーザン解析によって調べたところ、POR1の破壊株(Δpor1)においてはこれらの転写レベルは野生型株に比べ顕著に低下していた。また、PAT1のプロモーターのlacZによるレポーター解析では、Δpor1株ではオレイン酸による誘導は野生型株よりも抑制されていた。これらの結果から、Por1pが脂肪酸に応答する転写活性化因子であることを示唆している。

さらにPor1pとEGFPとの融合タンパク質を発現させ、Por1pが核に局在することを推定した。この融合タンパク質が脂肪酸存在下では核膜近傍で少数の強い輝点を形成したことを報告し、転写活性化との関係を示唆している。

また、欠失解析によりPAT1プロモーター上の-173から-166にあるCGAGCCGAの配列に注目し、これを変異配列に置き換えると、Δpor1株と同等レベルに脂肪酸による転写活性化が失われたことから、Por1pによる転写活性化はこの配列を介するものと推定したが、残念なことに大腸菌の高生産系ではPor1pの分解が激しく、Por1pの結合等を実証するには至らなかった。

第4章では、Δpor1株でなお脂肪酸含有培地における転写誘導が見られ、それがグルコース存在下で抑制されることから、S. cerevisiaeの非醗酵性炭素源による転写調節遺伝子ADR1のY. lipolyticaオルソログを検索し、C2H2 zinc finger タンパク質コードするYALI0D18678gを得ている。この遺伝子の破壊株ではオレイン酸あるいはn-デカンを含む培地におけるPAT1プロモーターの活性化の程度が低下し、POR1遺伝子との二重破壊株ではオレイン酸培地における誘導のさらなる低下が観察された。そこで、この遺伝子をCFU1 (Control of Fatty acid Utilization 1)と命名した。

以上、本研究はY. lipolyticaにおける脂肪酸に応答する遺伝子調節系を検討し、これに2つの転写因子が関わる証左を得ている。これらは菌類における脂溶性物質利用のための遺伝子調節システムに関する基礎的知見でもあり、学術的、応用的に貢献するところが少なくない。よって審査委員一同は、本論文が博士(農学)の学位論文として価値あるものと認めた。